Recent remarkable progress of molecular and biochemical researches on sugar chains has clarified some of sugar's important molecular and function and role, which makes it possible to develop pharmaceuticals sugar chains, and functional materials based on the sugar chains (oligosaccharides) possessing physiological activities. However, the oligosaccharides which are commercially available as reagents at present are limited to a few types and, to boot, very expensive. Moreover, such oligosaccharides are produced only on a reagent level and can not be supplied in large quantities.
Conventionally, oligosaccharides have been produced by such methods as extraction from natural substances, chemical synthesis, enzymatic synthesis, and combinations of these methods, but enzymatic synthesis has been considered best suited for their large-scale production as medicinal or functional materials.
This is because the enzymatic synthesis method is considered advantageous over the other methods in that (1) this method dose not require intricate steps for protection and deprotection such as necessary in the chemical synthesis method, and is also capable of quickly synthesizing the objective oligosaccharide, and (2) it is possible with this method to synthesize oligosaccharides having highly structural specificity because of substrate specificity of the enzyme used. Further, recent progress of biotechnology such as recombinant DNA technology have made it possible to mass-produce various types of enzyme economically, also contributing to establishing the superiority of enzymatic synthesis.
Two methods for the synthesis of oligosaccharides by enzymatic synthesis are available: (1) a reverse reaction of the hydrolase of an oligosaccharide is utilized, and (2) a glycosyltransferase is utilized. The former method has the advantage in that inexpensive monosaccharide can be used as substrate, but because it employs the reverse reaction to the hydrolysis, its practical application is very difficult in respects of yield of synthesis and applicability to the syntheses of oligosaccharides having a complicated structure.
On the other hand, the latter method, which utilizes a specific glycosyltransferase, is considered advantageous over the former method in that this method can be applied to the production of oligosaccharides having a complicated structure and is also high in yield of synthesis. Moreover, mass-production of various glycosyltransferases made possible by the recent progress of biotechnology such as recombinant DNA technology is contributing to the realization of practical application of said method.
However, sugar nucleotides, which are generally used as a sugar donor, are still expensive except for a few types thereof and actually supplied only in small amounts on reagent levels. For instance, regarding UDP-GalNAc which is a donor of N-acetylgalactosamine contained in the core portion of sugar chain of O-bound glycoprotein or sphingoglycolipid, there has been reported a method for synthesizing this compound from UDP-GlcNAc by using uridine diphosphate N-acetylglycosamine 4-epimerase (UDP-GlcNAc 4-epimerase) derived from animal tissue or Bacillus subtilis. (Analytical Biochemistry, 127, 171-177 (1982); J. Biol. Chem., 234(11), 2801-2805 (1959); JP-A-7-79792).
However, although UDP-GlcNAc is a sugar nucleotide which is relatively easy to prepare in large quantities. UDP-GlcNAc 4-epimerase exists only in small quantities in the animal tissues or bacterial cells. Also, there has been no report of preparation of this enzyme by recombinant DNA technology using an UDP-GlcNAc 4-epimerase gene. Thus, it has been practically difficult to produce UDP-GalNAc by making use of said enzyme, let alone bulk preparation of this enzyme itself.